Transition from normal tissue function to diseased tissue can be detected by quantifying irregular patterns. The degree of statistical similarities in a region of interest can carry valuable comparative information about the structural features of the tissue and can help to characterize tissue, i.e., analyze disease localization and progression. To visualize subsurface structural features of biological tissues, we have developed a user-friendly polarization imaging system that simultaneously images cross- and co-polarized light. We have developed a quantitative statistical tool, based on Pearson correlation coefficient analysis to enhance the image quality and reveal regions of high statistical similarities within the noisy tissue images. We have shown that under certain conditions, such maps of the correlation coefficient are determined by the textural character of tissues and not the choice of the reference image region, providing information on tissue structure. As an example, the subsurface texture of a demineralized tooth sample was enhanced from a noisy polarized light image. We have recently adapted the polarized camera to be used in a colposcopeto study tissue structures in the cervix. Fluorophore lifetime imaging is a promising tool for studying tissue environment such as tumors. The lifetime (time for an electron to return from excited state to initial state) of a fluorophore can vary in response to changes in the immediate environment such as temperature, pH, tissue oxygen content, nutrient supply, and bioenergetic status. Mapping the lifetime and location of a fluorophore in tissue at different depths can be used to monitor such parameters. Toward this goal, we have developed a time-resolved lifetime imaging system for in vivo small animal studies that maps fluorophores lifetimes. The system consists of a single source-multiple detector array that scans the surface of the tissue. Using several source-detector separations, one is able to probe different depths of the medium. We have used a pH sensitive dye in the near-infrared region in order to study the tumor environment below the skin. We have demonstrated that by using simplified back projections we are able to map near surface fluorescent lifetime in vivo. Combining this with the pre-calibrated lifetime response to pH, we have shown that biologically plausible, non-invasive, quantification of pH in mouse tumors can be determined. In collaboration with Dr. Capala in the Radiation Oncology Branch project of NCI who has developed probe for in vivo monitoring of HER2-positive cancer (for example breast cancer) non invasively and HER2 specific delivery of therapeutic agents. We have been able to quantify the bio-distribution of the probe and, HER2 receptors on tumor cells, showing the potentila of this method for monitoring non-invasively and quantitatively the teatment response. To further this work to include deeper tissue imaging we have examined the use of novel data-types for reconstructing location and lifetime of fluorophores embedded deeper in a turbid medium. We have developed a set of local data-types which provide enhanced noise tolerance over the standard global data-types for imaging purposes. Analysis of deeply embedded tissue abnormalities, using time-resolved fluorescence, should take into account highly scattering nature of biological tissues. To address corresponding complications we investigated the limitations of previously developed analytical model of photon migration for localized fluorophores. It was shown that to better describe experimental results the model should incorporate more realistic distribution of fluorescence lifetimes thus providing more flexibility to the inverse model to converge. This analytic model has been used as a forward model to reconstruct the lifetime and location of a point source fluorophore. Other advances have been made in studying the noise sensitivity of different data-types in time-resolved fluorescence imaging, we suggested a new local set of data-types that is likely toprovide more stability to noise than classical statistical (global) data-types used in diffuse optical tomography. We have pursued also another approach to quantification of fluorescence lifetimes of deeply embedded fluorophores that can work when intrinsic lifetime of the fluorophore is comparable to photon migration time in the medium, using general scaling relations to correct observed time-resolved intensity distributions from fluorescent targets at a given depth z inside turbid medium to an expected surface distribution (from the same fluorophore), revealing the intrinsic fluorescence lifetime without the need for full-scale reconstruction. Similar corrections could be applied when comparing the time-resolved data obtained from the same deeply embedded fluorophore by several detectors, positioned at different distances from the source (excitation photon entry point into the medium). . We experimentally verified these relations, usingtissue-like phantoms. Developed random walk model of time-resolved fluorescence imaging substantiates these scaling relations. Another optical imaging modality of interest to the group is Two-Photon Microscopy. Colleagues at NHLBI have recently developed a new system for Two-Photon imaging based on the Total Emission Detection (TED) principle. Here instead of using a standard TED system where only transmitted or reflected light is collected the TED Two Photon system captures all light emitted from the sample. Researchers in this group were contacted to model Two Photon emission and evaluate the systems performance. Using our Monte- Carlo simulation code we were able to evaluate different aspects of Two-Photon Imaging and the related field of Fluorescence Correlation Spectroscopy in collaboration with Section on Cell Biophysics at NICHD. The oncology community is testing a number of novel targeted approaches for use against a variety of cancers. With regard to monitoring vasculature, it is desirable to develop and assess noninvasive and quantitative techniques that can not only monitor structural changes, but can also assess the functional characteristics or the metabolic status of the tumor. We are testing three potential noninvasive imaging techniques to monitor patients undergoing an experimental therapy: infrared thermal imaging (thermography), laser Doppler imaging (LDI) and multi-spectral imaging. These imaging techniques are being tested on subjects with Kaposi s sarcoma (KS), a highly vascular tumor that occurs frequently among people infected with acquired immunodeficiency syndrome (AIDS). Cutaneous KS lesions are easily accessible for noninvasive techniques that involve imaging of tumor vasculature, and they thus represent a tumor model in which to assess certain parameters of angiogenesis. The KS studies are ongoing clinical trials under four different NCI protocols. We have shown that our multi-modality techniques can non-invasively monitor the functional properties of the tumor and surrounding tissues and has the potential to predict treatment outcomes. To better quantify the blood volume and oxygenation we have developed a frequency domain Optical Coherence Tomography system to better correlate the strctural information with functional activities of the tumor. Integration of this technique is underway. High-resolution confocal laser microscopy is an intensively active field in modern bioimaging technologies because this technique provides sharp, high-magnification, three dimensional imaging with submicron resolution by non-invasive optical sectioning and rejection of out-of-focus information. We have developed a simple fiber-optic confocal microscope with nanoscale depth resolution beyond the diffraction barrier. We are extensively working to increase the acquisition time for data collection in 3D.